Pipetting, contamination....

ChenHA via methods%40net.bio.net (by hzhen from freeuk.com)
Fri Feb 23 05:58:30 EST 2007

DK wrote:
> In article <1172181256.088647.198980 from a75g2000cwd.googlegroups.com>, "peter" <peter.ianakiev from gmail.com> wrote:
>>> Jeez, I've done this experiment yesterday:
>>> 1. Took a 1 ml Pipetman, set it to 1 ml and pipetted overnight E.coli
>>> culture up and down 20 times.
>>> 2. Immediately after that, changed a tip, took a bottle of sterile
>>> Terrific Broth, pipetted 20 times, set at 37C for 20 hours.
>>> The result: no signs of bacterial growth today ==> no aerosol
>>> contamination.
>>> DK
>> Seems like a good experiment, I just wonder what volume of the TB
>> media you used?
> The standard square bottle filled with 100 ml. I still have that bottle,
> so if you have your doubts, I can look at it very attentively by Monday. 
> But, you know, E.coli can grow anaerobically so shaking is not 
> strictly necessary...
> I pretty much knew the outcome based on the fact that I am still 
> using the same ~1.5 years old bottle of SOC used for an umpteen 
> (more like ~ 100 :-)) 

You must be more careful than I am.  I rarely reuse media bottles or 
flask once they have been opened.  I know that they often stay clear for 
days after being opened, but still, I don't like taking chances.

I think a lot of this has to do with the environment (perhaps the air 
quality) you work in.  I have worked in labs where I tried to be 
extremely careful, but still strange colourful colonies grew on plates 
after it's left on bench for some time.  In other labs I rarely see this 
even though I was less careful.

> electroporations, each involving the same 
> 1 ml Pipetman and no filter tips all along. Amazingly, still no visible 
> growth in the bottle and, as recently as two days ago, 20 out of 20 
> clones analyzed contained the expected plasmid. 
> DK
> P.S. This topic reminded me of one curious observation: 
> Everyone I know who does "microbiology" outside of sterile hood 
> is engaged in the practice of flaming necks of every bottle and 
> flask they use. When asked why they are doing it, the only answer 
> ever given is "to keep it sterile". Problem is, for as far as I can tell,
> the way it is done, it's a completely useless exercise in at least 
> 9 out of 10 times. 

I don't flame anything, and don't understand why people insist on 
flaming.  Flame in any case seems to cause more problems, for example, 
I have seen on more than one occasions where flame cause the plastic 
gloves people wear to go up in flame as well (spreader dipped in 
ethanol, flamed, flaming ethanol dripped onto gloves, whooomp!)


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